Tyrosine phosphatase activity is restricted by basic charge substituting mutation of substrates

Phosphorylation controls important cellular signals and its dysregulation leads to disease. While most phospho-regulation studies are focused on kinases, phosphatases are comparatively overlooked. Combining peptide arrays with SAMDI mass spectrometry, we show that tyrosine phosphatase activity is restricted by basic amino acids adjacent to phosphotyrosines. We validate this model using two β-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K). These mutants introduce a basic residue next to Y654, an established phosphorylation site where modification shifts β-catenin from cell–cell adhesions and towards its essential nuclear role as Wnt-signaling effector. We show that T653-basic mutant β-catenins are less efficiently dephosphorylated by phosphatases, leading to sustained Y654 phosphorylation and elevated Wnt signals, similar to those observed for Y654E phospho-mimic mutant mice. This model rationalizes how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6000+ potentially relevant cancer mutations.

www.nature.com/scientificreports/ We reasoned that the higher phosphorylation level of Y654 in the mutants is due to decreased phosphatase activity, and which would lead to elevated Wnt signals and associated disease. Below, we demonstrate that introduction of a basic residue adjacent to Y654 in β-cat antagonizes its dephosphorylation by SHP1 in HEK cells, favoring persistent phosphorylation and elevation of AXIN2, which is a canonical β-cat target in the Wnt pathway 32 . This mechanism rationalizes neuro-developmental phenotypic similarities between T653K Bfc and Y654E phospho-mimic mutant mice 33 , as well as how β-cat missense mutations proximal to Y654 may drive epithelial cancers.

Results
Database search identifies 6000+ relevant cancer mutants. To identify cancer mutants with basic residue substitutions adjacent to phosphotyrosines, we employed the protein post-translational modification (PTM) database PhosphoSitePlus and the single-nucleotide variations (SNV) cancer database BioMuta 28,29 . We used PhosphoSitePlus to identify all reported phosphotyrosine sites and selected for those that had corresponding cancer mutations at their + 1 and − 1 positions in BioMuta. We then filtered these sites to select those with a R or K amino acid in either the wildtype or mutant forms, to identify base-substitution missense cancer mutations including both addition and removal of basic residues. Some notable sites identified are listed in Fig. 2c and a comprehensive list of the database search results can be found in SI Table 1. To our surprise, this search identified more than 6000 mutants, though most had an unclear contribution to protein function. While these pY-proximal missense mutations occur frequently, how they impact phosphatase activity (as opposed to kinase activity) is poorly understood.
We selected the CTNNB1 gene/β-cat protein for further study, because T653R and T653K are proximal to the well-studied Y654 phospho-site and are associated with reduced cell-cell adhesion and elevated nuclear signaling. Moreover, β-cat T653K was also identified in a forward genetic screen for mutations affecting craniofacial development in mice, generating phenotypes that overlap with dominant human β-cat/CTNNB1 mutations causing intellectual disability with similar syndromic features 30 .
As a physical link between cell surface cadherins and the actin-binding protein α-catenin, β-cat plays a critical role in cell-cell cohesion. Outside of the cadherin-catenin complex, β-cat also functions as an essential effector of the Wnt pathway, pairing with DNA-binding factors to activate transcription of genes that drive cell fate decisions 34 . The degree to which β-cat participates in these two functions is determined by the availability and relative affinity of β-cat binding with its competing partners 35 . One modification that shifts β-cat from its cellcell adhesive function in favor of nuclear signaling is its phosphorylation at Y654 by Src family kinases (SFKs) where this modification reduces β-cat binding to the cadherin cytoplasmic domain 36,37 and also antagonizes its degradation by the β-cat phospho-destruction complex 38,39 , leading to additional modifications that enhance transcriptional activity 33 . Below, we sought to determine whether β-cat basic T653K/R mutants lead to enhanced Wnt signaling through persistent phosphorylation of Y654 due to lack of PTP activity. β-cat T653 basic mutants show greater phosphorylation at Y654. We obtained a FLAG-β-cat / pcDNA3 plasmid 40 and generated two basic mutants T653R, T653K and the neutral mutant T653A as control. All four constructs expressed similarly in transfected HEK 293T cells. Consistent with previous studies 39 , no Y654 phosphorylation was observed in these cells using an anti-β-cat pY654 site-specific phosphoantibody (Fig. 3a). Co-transfection with constitutively active Src kinase (Src/CA) led to robust detection of WT β-cat with the β-cat-pY654 specific antibody (Fig. 3b). Unfortunately, this antibody was unable to distinguish phosphorylation of our mutant β-cats where the adjacent T653-residue was altered, because the epitope includes adjacent residues (A-T-pY-A-A). We next repeated this experiment with a general phospho-tyrosine antibody (clone PY20). We found, however, that even a pan-pY antibody recognized the WT and T653A mutant β-cat proteins better than the T653K/R basic mutants (Fig. 3c), consistent with evidence that the PY20 antibody also disfavors substrates with basic residues adjacent to pY 41 . Thus, even the pan-phosphotyrosine antibody, PY20 will underreport the pY abundance for these protein sequences.
While the pan-PY20 antibody fails to recognize all pY sequences equally, it does have sufficient affinity for each of the β-cat constructs. Hence, we expected that an excess of the PY20 antibody could quantitatively immunoprecipitate the four phosphorylated constructs (including exogenously expressed FLAG-tagged β-cats). Subsequent immunoblotting for the FLAG epitope then gives β-cat pY status (Fig. 3d). With this approach, we found that the β-cat T653K mutant showed significantly (p = 0.0120, t-test) more phosphorylation by Src/CA than did WT; T653R mutant also exhibited slightly more phosphorylation than did WT and T653A, consistent with our in vitro peptide library profiling results (Fig. 3e). To explore the role of phosphatase regulation of this site, we co-transfected SHP1, a known PTP to dephosphorylate β-cat pY654 39 , along with β-cat constructs and Src/CA. We found that the amount of phospho-β-cat was reduced by roughly threefold, suggesting β-cat-pY654 is a possible target of SHP1 in vivo (Fig. 3f). Importantly, treatment with the pan-tyrosine phosphatase inhibitor, sodium pervanadate (Na 3 VO 4 ) eliminated differences in phosphorylation between WT and mutant β-cat proteins (Fig. 3g). This data demonstrates that β-cat with a basic residue adjacent to pY results in higher phosphorylation levels at pY654, and that inhibition of PTPs eliminates the observed differences in phosphorylation levels. These data strongly suggest that differential phosphatase activity (not necessarily SHP1) towards β-cat pY654 versus WT β-cat can explain differences in phospho-abundance, where PTP activity is restricted by basic residues adjacent to the pY site. We cannot exclude the possibility that some of the SHP1-mediated reduction in β-cat pY654 phosphorylation in cells may be indirect, for example through inhibition of Src/CA signaling via Y419 42 .
β-cat basic charge mutants manifest enhanced Wnt-signaling. To gain additional evidence for increased phosphorylation levels in the β-cat T653K/R mutants, and to address how the β-cat T653K/R mutants impact Wnt-signaling, we analyzed AXIN2 levels in western blots as a downstream reporter. This protein is a universal target gene and negative-feedback regulator of the pathway 32 . We used the CRISPR-Cas9 strategy to generate a HEK β-cat KO cell line (See SI for the generation and characterization of the knockout cells) and selected clones lacking AXIN2 induction upon treatment with the Wnt pathway agonist and GSK3 inhibitor, LiCl (Fig. 4a, clone 16) 32 . We transfected FLAG-tagged constructs into the β-cat KO  www.nature.com/scientificreports/ depleted after transfected cells were serum starved (Fig. 4b, first lane) and increased only after cells were supplied with FBS, allowing us to effectively synchronize our cells.
Using this method, we found that the T653R and T653K mutants expressed significantly more AXIN2 than WT ( Fig. 4b; p = 0.0308 and 0.0301, t-test); the changes in AXIN2 expression in T653A mutant were not significant compared to WT (p = 0.3732, t-test). These findings are consistent with the elevated pY654 phosphorylation observed in Fig. 3e, and demonstrate that the more phosphorylated basic mutants contribute to elevated β-cat signaling. To show that phosphatase activity was required for the different levels of β-cat signaling, we treated cells with 100 μM Na 3 VO 4 to inhibit PTPs and found that WT β-cat induced AXIN2 to similar levels as the T653K/R mutants (Fig. 4c). Together, these experiments establish that disease-associated missense mutant forms of β-cat (T653K/R) display enhanced Wnt-signaling activity through a positive-charge gating (or steric hindrance) mechanism that restricts tyrosine phosphatase substrate selectivity at pY654.

Discussion
This paper shows how basic residues adjacent to pY sites in proteins can antagonize dephosphorylation and result in higher phosphoprotein levels, and lead to corresponding impacts in signaling pathways. We identified β-cat as a relevant example, because T→R/K mutations are associated with disease but remain poorly understood. Our work shows that both basic mutants have higher tyrosine phosphorylation levels and lead to higher levels of AXIN2 in cells, consistent with elevated Wnt signaling compared to WT β-cat (Fig. 5). Importantly, this difference is eliminated when cells are treated with a phosphatase inhibitor, suggesting that phosphatases differentially target mutant versus WT β-cat. This work offers an alternate explanation for neuro-developmental phenotypic  (1 mg) were immunoprecipitated with a FLAG antibody. Pan-phosphotyrosine antibody displays different binding affinity to WT and mutants.(d) β-cats were immunoprecipitated with pY and blotted for FLAG to quantify their Y654 phosphorylation. (e) pY654 is quantified by first taking the ratio of FLAG signal in pY-pulldown and the lysate input to account for loading/expression differences. Then, the corrected intensities of mutants were compared to WT. In biological triplicate experiments, both basic β-cat T653 mutants are more phosphorylated than WT. Error bars: one standard deviation. *: p < 0.05 in t-test. (f) β-cat pY654 is a substrate for SHP1. (g) Inhibiting PTPs with pervanadate (100 μM) eliminates the difference in Y654 phosphorylation between WT and mutants. www.nature.com/scientificreports/ similarities between Y654E phospho-mimic and T653K bfc mutant mice 30,33 . The less efficient dephosphorylation of pY654 with an adjacent basic residue favors persistent phosphorylation and elevation of β-cat-mediated gene expression, rationalizing its resemblance to a permanent phospho-mimic in the developmental process. Our study also points to a potential SHP1 activation treatment strategy through induction of SHP1 expression or small molecule activators 43 . Assays of phosphatase activity are very challenging, and largely not well-suited to direct determine phosphatase specificity. Traditional assays frequently use generic and non-specific small molecule substrates such as para-nitrophenylphosphate (pNPP) and 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), which do not provide sequence context 44,45 . One approach described by Cesareni and co-workers used peptide arrays but required the use of a substrate-trapping mutant that may not retain the substrate specificity of the wild type enzyme 14 . In a combinatorial approach using libraries of phosphopeptides immobilized to beads, Pei and coworkers identified peptides that were active substrates for the PTPs 24-26 . However, the use of a tyrosinase to label the active beads could impart its own sequence specificity and the combinatorial approaches do not provide quantitative results for every peptide sequence in the library. For example, fewer than one percent of the peptides were analyzed, giving an incomplete understanding of the enzyme specificity. SAMDI-MS is a label-free and high throughput assay that directly address these issues and provides a more complete assessment of the PTP specificity.
These benefits of the label-free assay allowed us to use SAMDI-MS to analyze peptide arrays and revealed general rules for the sequence dependent activity of phosphopeptide substrates. Remarkably, the rule we describewhere PTPs disfavor basic residues proximal to pY-is surprisingly general. In this work, we show how this insight can explain signaling in β-cat mutants and in previous work we addressed its role in insulin receptor 22 . We note that our database search identified over 6000 relevant cancer mutations that could employ this missense mutation/PTP restriction mechanism, and suggests that this mechanism can have a broad relevance in understanding mutations that underlie cancers.
This work demonstrates that disease-related mutants can operate by altering phosphoprotein levels through a modulation of phosphatase activity. The study is also significant because the underlying hypothesis was revealed through biochemical studies of PTP specificity, which was enabled by the development of a robust label-free assay. We expect that reports of mechanistic roles for phosphatase-directed phenotypes will become more common and lead to new diagnostic markers and therapeutic targets in drug discovery.

Methods
Database search. Phosphotyrosine sites were downloaded from PhosphoSitePlus and single-nucleotide variations (SNVs) in cancer were downloaded from BioMuta 28,29 . The cancer mutations at + 1 and − 1 positions to a phosphotyrosine that added or removed a basic residue (R or K) were extracted. A full list of identified gene sites and local sequences can be found in Table S1 and S2.  www.nature.com/scientificreports/ Immunoblotting. Lysates or IP eluates were resolved by 4-20% Criterion TGX Midi gels (Bio-Rad) and transferred to nitrocellulose membranes (GE) following the manufacturer's protocol. Membranes were blocked in Intercept (TBS) blocking buffer (LI-COR) for 1 h at room temperature. Membranes were incubated overnight with primary antibodies at 4 °C and then with fluorophore-conjugated secondary antibodies (LI-COR) for 1 h at room temperature. All antibodies were used at the dilutions suggested by the manufacturers. The bands were visualized by LI-COR Odyssey Fc Imaging System. The images were processed with ImageJ. RNAs were reconstituted and diluted to 5 μM with duplex buffer; Cas9 protein to 10 μM with PBS. HEK cells were plated in 12-well plates at a seeding density of 1.0 × 10 5 cells in 1 mL DMEM-complete a day before. For one reaction, sgRNA (20 μL), Cas9 (15 μL), DMEM (30 μL, no serum and antibiotics) and Lipofectamine RNAiMax (4 μL, Invitrogen) were mixed and incubated at room temperature for 20 min. The complex was treated to the cells in complete medium and incubated overnight at 37 °C and 5% CO 2 . The medium was changed and the cells were allowed to recover for 1 day. Cells were split to maintain 30-50% confluency. The sgRNA-Cas9 treatment was repeated 3 times. Cells were expanded and sorted with a flow cytometer (FACSMelody, BD) to 96-well plates to grow single cell colonies. The colonies were screened for low (< 5 ng/well at confluent) β-cat expression using a β-cat ELISA kit (Invitrogen). The selected colonies were verified with western blot. The knockout cell line was maintained in the same conditions as WT.

Data availability
The datasets analyzed during the current study are available in the PhosphoSitePlus (https:// www. phosp hosite. org/) and BioMuta (https:// hive. bioch emist ry. gwu. edu/ biomu ta/) repositories. The data for β-catenin is available using Uniprot accession code P35222 in both databases. All data generated during this study are included in this published article (and its Supplementary Information files).